A rapid method to monitor repair and mis-repair of DNA double-strand breaks by using cell extracts of the yeast Saccharomyces cerevisiae.
We present a rapid in vitro method to scan the repair of DNA double-strand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52Delta) strain of the yeast Saccharomyces cerevisiae. The fidelity of rejoining was determined by the expression of the lacZ gene after bacterial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-type and mutant strains to form circular plasmids with almost equal efficiency. However, the fidelity of rejoining was lower for the rad52Delta extract than for normal wild-type.
Jha
B
B
Department of Biotechnology, L. N. Mithila University, Darbhanga-846 008, India.
Ahne
F
F
Kistler
M
M
Klaus
C
C
Eckardt-Schupp
F
F
D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013347:Subcellular Fractions / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA